One type of widely used electrophoresis is isoelectric focussing, wherein substances, such as proteins, are separated according to their pI-value. For isoelectric focussing, sample loading has traditionally been performed by cup loading by placing a cup on the gel and letting a sample pass through the cup into the gel. The cup is positioned on the gel for the whole electrophoresis run.
Alternatively for dried gels, the sample may be mixed with electrophoresis buffer and used as a rehydration solution to rehydrate the dried gel, such as Immobiline DRYSTRIP™ gels.
More recently, sample application paper in the form of conventional filter paper, has been placed between the electrode and the electrophoresis gel to load a sample into an electrophoretic gel. This functions satisfactorily for sample application from the anode side of the gel. However, this approach does not work when using acidic pH intervals extending to pH-values bellow pH 4, for example a pH 3-11 strip, a pH 3-7 or a pH 3-5 strip. When samples are paper loaded from the anodic (acidic) side of the IPG strip, the proteins and peptides will be retained in the paper
As an alternative, rehydration loading can be used in these pH intervals. However, rehydration loading is not possible with swollen gels, such as pre-swollen RTG (ready-to-go) strips. Thus, these kind of gels need an alternative loading, especially for application of large samples which is very difficult today.
The co-pending application WO 05/062032 provides an alternative way to load samples onto electrophoretic IPG gels which avoids the problem with protein precipitation.
This invention enables sample loading from the cathode side of the IPG gel or strip. According to this invention, the sample is applied to an acidic interval IPG gel or strip, such as a RTG (ready-to-go) strip. This novel application enables sample loading in preparative amounts of protein. This is achieved by providing use of a positively charged support for sample application from the cathode side of the gel.
In spite of this improvement over prior art, there is still a need of alternative ways of sample loading onto electrophoretic gels.